This Section has focused on the role of Ca++ as a regulator of gene expression and cellular physiology. Using CAI as a tool, we have investigated Ca++-regulated molecular events. Several clones were identified from a subtractive hybridization of CAI-resistant v. wild type A2058 human melanoma cells. CAIR-1 is a unique 2.8kb about 62kDa cytoplasmic protein. It is overexpressed 2-4 fold in resistant cells not amplified; expression is not increased with short term exposure to CAI. CAIR-1 is expressed ubiquitously in tissue, with phylogenetic expression limited to higher eukaryotes. The expressed native protein is about 76kDa; the size difference is attributed in part to basal phosphorylation, documented by in vivo phosphorylation assays. CAIR-1 has both CKII and protein kinase C phosphorylation sites, and a single tyrosine phosphorylation site. It has an N-terminal leucine zipper and a proline-rich domain. Our studies suggest that CAIR-1 is a substrate for a PMA-insensitive protein kinase C. Further characterization of structure and function are ongoing. A second approach to the effect of Ca++ on gene regulation has used CAI as a tool to differentiate between a requirement for increased intracellular Ca++ and the source: intracellular release v. influx. Fos has long been known to require Ca++, but our recent collaborative studies have indicated that either influx or intracellular release may provide Ca++ to induce fos expression, depending upon the specific ligand. In RAT-1 cells expressing a fos-CAT construct, endothelin-1 can induce expression through either influx or intracellular release, however, EGF induction requires influx. Similarly, expression of matrix metalloproteinase-1 and -2 expression is inhibited by CAI. Fos is involved in MMP-1 transcription though AP-1 complex; the Ca++-sensitive transcription factor for MMP-2 has not been defined. We hypothesize that Ca++ balance in the cell may be altered by increasing Ca++ channel number. The only identified and cloned nonvoltage-gated Ca++ channel is the human homolog of trp, a Drosophila photoreceptor protein. Collaboratively, we are attempting to express this htrp homolog in both normal and malignant cells to determine how overexpression alters Ca++ homeostasis, and transformed and metastatic phenotypes. The htrp genes cloned are from normal human brain cDNA libraries and expression is restricted to brain. Homology is low between brain htrp genes, suggesting a family of related but independent trp genes. Cloning of a human trp homolog from breast and ovarian cancer cells is ongoing.